Transcriptome analysis reveals the regulation of miR-19b on inflammation in bovine mammary epithelial cells.

MicroRNAs (miRNAs) are involved in various biological processes where they regulate the expression of mRNAs. Bovine mammary epithelial cells (bMECs) are functional cells that mediate mammary inflammatory immunity. Although numerous miRNAs regulate the function of bMECs, the role of miR-19b in bMECs has not been reported. In this study, the transcriptome of miR-19b overexpressed bMECs was analyzed by RNA-seq. Additionally, the differentially expressed genes (DEGs) were analyzed to establish the role of miR-19b in bMECs. The results revealed 269 DEGs between the miR-19b overexpression group and the negative control, including 199 up-regulated and 70 down-regulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that the DEGs regulated immune and inflammatory responses through Staphylococcus aureus (S. aureus) infection and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In addition, the expression of miR-19b was significantly upregulated in lipophosphoric acid (LTA)-induced bMECs, and overexpression of miR-19b negatively regulated the expression of inflammatory cytokines IL-1ß and IL-6, thereby alleviating the inflammatory response of LTA-induced bMECs. Based on the above results, we speculate that miR-19b may inhibit in dairy cow mammary inflammation caused by S. aureus, and this process may be mediated through the regulation of relevant gene expression and signaling pathways. The findings from this study provide a new reference for analyzing the molecular regulation of miR-19b in bMECs.

The role of microRNAs in the regulation of critical genes and signalling pathways that determine endometrial receptivity.

Endometrial receptivity is the ability of the endometrium to accept embryos. Thus, endometrial receptivity dysfunction is an important factor leading to embryo implantation failure. A good endometrial receptivity provides a suitable environment for embryo implantation, improving the embryo implantation rate. The "implantation window" stage, or the receptive stage of the endometrium, is regulated by various hormones, genes, proteins and cytokines, among which microRNAs (miRNAs) and their target genes have a regulatory effect on endometrial receptivity. This review outlines the relationship between endometrial receptivity and pregnancy, the mRNAs and related signalling pathways that regulate endometrial receptivity, and the regulatory role of miRNA in endometrial receptivity, providing a deeper understanding of the regulatory mechanisms of miRNA on endometrial receptivity in humans and animals and reference for the endometrial receptivity-related research.

Research progress of intramuscular fat formation based on co-culture.

Intramuscular fat (IMF) is closely related to the meat quality of livestock and poultry. As a new cell culture technique in vitro, cell co-culture has been gradually applied to the related research of IMF formation because it can simulate the changes of microenvironment in vivo during the process of IMF cell formation. In the co-culture model, in addition to studying the effects of skeletal muscle cells on the proliferation and differentiation of IMF, we can also consider the role of many secretion factors in the formation of IMF, thus making the cell research in vitro closer to the real level in vivo. This paper reviewed the generation and origin of IMF, summarized the existing co-culture methods and systems, and discussed the advantages and disadvantages of each method as well as the challenges faced in the establishment of the system, with emphasis on the current status of research on the formation of IMF for human and animal based on co-culture technology.

Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle.

BACKGROUND: Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. RESULTS: We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. CONCLUSION: In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

ncRNAs regulate bovine adipose tissue deposition.

Lipid metabolism, which encompasses synthesis and degradation of lipids, is critical for a wide range of cellular functions, including structural and morphological properties of organelles, energy storage, signalling, and the stability and function of membrane proteins. Adipose tissue is a dynamic tissue type that performs a lot of significant physiological functions, including secretion, and is involved in maintaining homeostasis and in regulatory roles of other tissues based on paracrine or endocrine. More recently, several classes of non-coding RNAs (ncRNAs), such as long non-coding RNA (lncRNA), microRNA (miRNA) and circular RNA (circRNA), have been discovered in adipocytes, and they act as critical regulators of gene expression in adipogenesis and regulate adipogenesis through multiple pathways. In the present paper, we discussed several classes of non-coding RNAs and summarized the latest research on the regulatory role of ncRNAs in bovine adipogenesis. We gave examples for known modes of action to look forward to providing reference information future scientific research in cattle breeding.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene.

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3' untranslated region (3' UTR) of the NKIRAS2, but not the 3'UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

Characterization of the promoter region of the bovine IRX3 gene: roles of SREBF2 and PPARG.

As a member of the Iroquois homeobox gene family, the IRX3 gene plays an important role in regulating the growth, development and fat deposition of chordates. In the present study, we found, using real-time PCR, that the bovine IRX3 gene was highly expressed in lung, kidney, heart, subcutaneous fat and longissimus dorsi muscle. We cloned the full-length sequence of the bovine IRX3 gene promoter and constructed eight series of 5' deletion promoter plasmid luciferase reporter assays and then transfected them to 3T3-L1 and C2C12 cell lines to detect its core promoter regions. The results showed that the core promoter of bovine IRX3 was located within a -292/-42 bp region relative to the transcriptional start site. Furthermore, sequence analysis identified eight CpG islands in the promoter region. A chromatin immunoprecipitation assay in combination with site-directed mutation and siRNA interference demonstrated that SREBF2 and PPARG binding occurs in region -292/-42 and is essential in bovine IRX3 transcription. These results lay an important theoretical foundation for exploring the molecular regulation mechanism of the IRX3 gene in bovine fat deposition.

MicroRNA-214 regulates immunity-related genes in bovine mammary epithelial cells by targeting NFATc3 and TRAF3.

In human, microRNA-214 (miR-214) plays crucial roles in mechanisms of immunity. However, the potential importance of miR-214 in immune mechanisms in dairy cows has not been investigated. In this study, we assessed potential immunity-related functions of miR-214 in human 293A cells and in bovine mammary epithelial cells (BMECs). We found that NFATc3 and TRAF3 could be targeted by miR-214 in both 293A cells and BMECs. We also found that miR-214 indirectly inhibited the expression of MAP3K14, TBK1 and inflammatory cytokines IL-6 and IL-1ß. Taken together, our data revealed miR-214 regulated immunity-related genes by targeting NFATc3 and TRAF3, which provides insight into the molecular basis of immunity.

Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene.

It has been reported previously that bovine miR-146a (bta-miR-146a) is significantly differentially expressed in mammary glands infected with mastitis, compared with healthy udders. This suggests that bta-miR-146a plays an important role in the regulation of mammary inflammation. However, the specifics of this function have yet to be elucidated. Bovine mammary epithelial cells (bMEC) represent the first line of defense against pathogens and have important roles in initiating and regulating inflammatory responses and innate immunity during infection. In this study, a double luciferase reporter assay was used to confirm that bta-miR-146a directly targets the 3' UTR of the tumor-necrosis factor receptor-associated factor 6 (TRAF6) gene. To elucidate the role of bta-miR-146a in innate immune responses, either a mimic or inhibitor of bta-miR-146a was transfected into bMEC stimulated with lipopolysaccharide, which activates the innate immune response through the toll-like receptor (TLR) 4/nuclear factor (NF)-κB signaling pathway. Forty-eight hours posttransfection, quantitative real-time PCR and Western blots were used to detect the expressions of the related genes and proteins, respectively. An ELISA was used to measure the quantity of inflammatory factors in culture supernatants. The results showed that bta-miR-146a significantly inhibits both mRNA and protein expression levels of bovine TRAF6, and ultimately suppresses downstream expression of NF-κB mRNA and protein. As a result, production of NF-κB-dependent inflammatory mediators such as tumor necrosis factor α, IL-6, and IL-8 are suppressed following lipopolysaccharide stimulation of bMEC. Thus, we concluded that bta-miR-146a acts as a negative feedback regulator of bovine inflammation and innate immunity through downregulation of the TLR4/TRAF6/NF-κB pathway. This study presents a potential regulatory mechanism of bta-miR-146a on immune responses in bovine mammary infection and may provide a potential therapeutic target for mastitis.

LncRNA HULIB promotes LPS induced inflammatory response in bovine mammary epithelial cells via PP2AB.

Bovine mastitis is regulated by genetic and environmental factors. Long non-coding RNAs (LncRNAs), which regulate various biological processes (immune system and biological development), have been found to play a role in bovine mammary inflammation responses. Here, a novel functional lncRNA, named lncRNA HULIB, was identified as a regulator during bovine mastitis. qRT-PCR and subcellular fractionation assays showed that lncRNA HULIB was significantly up-regulated in LPS-induced bMECs and was mainly localized in the cytoplasm. Gain- or loss-of-function experiments demonstrated that an increase in lncRNA HULIB expression elevated the expression of TLR4 and NF-κB1, which enhanced NF-κB activity, promoting the expression of pro-inflammatory cytokines (IL-6, IL-8, IL-1ß, etc) and apoptosis-related genes (BAX, CASP9 and CASP3, etc), while the expression of proliferation-related genes (PCNA, Cyclin D1, Cyclin D2, CDK4 and CDK2) was down-regulated. Ultimately, these changes exacerbated the LPS-induced inflammatory response. Mechanistically, RNA pull-down and RNA immunoprecipitation (RIP) assays revealed that lncRNA HULIB could directly bind the PP2AB protein to regulate inflammatory responses. Overall, lncRNA HULIB is a pro-inflammatory regulator, and its silencing can alleviate the inflammatory responses of bMECs, providing a potential strategy for molecular therapy of bovine mastitis.

LncRNA CA12-AS1 targets miR-133a to promote LPS-induced inflammatory response in bovine mammary epithelial cells.

Bovine mastitis seriously affects milk production and quality and causes huge economic losses in the dairy industry. Recent studies have shown that long non-coding RNAs (lncRNAs) may regulate bovine mastitis. In this study, the expression of lncRNA CA12-AS1 was significantly upregulated in LPS-induced bovine mammary epithelial cells (bMECs) but negatively correlated with the expression of miR-133a, suggesting that it may be related to the inflammatory response in bMECs. Dual luciferase reporter gene assay revealed that miR-133a is a downstream target gene of lncRNA CA12-AS1. Furthermore, lncRNA CA12-AS1 silencing negatively regulated the expression of miR-133a inhibited the secretion of inflammatory factors (IL-6, IL-8 and IL-1ß) and decreased the mRNA expression levels of nuclear factor kappa B (NF-κB) (p65/p50) and apoptosis-related genes (BAX, caspase3 and caspase9). LncRNA CA12-AS1 silencing also promoted the mRNA expression levels of the Tight junction (TJ) signaling pathway-related genes (Claudin-1, Occludin and ZO-1), apoptotic gene BCL2, proliferation-related genes (CDK2, CDK4 and PCNA) and the viability of bMECs. However, overexpression of lncRNA CA12-AS1 reversed the above effects. These results revealed that lncRNA CA12-AS1 is a pro-inflammatory regulator, and its silencing can alleviate bovine mastitis by targeting miR-133a, providing a novel strategy for molecular therapy of cow mastitis.

RNA Sequencing Reveals the Involvement of Serum Exosomal miRNAs in Early Pregnancy in Cattle.

Low fertility is the main cause of the low productivity in beef cattle and is mainly associated with a lack of conception after fertilization. The establishment of early pregnancy in cattle is a complex physiological process, and embryo implantation is crucial for the successful establishment of pregnancy. Exosomal miRNAs play an important role in regulating mammalian embryo implantation and development. This study used synchronous estrus technology to extract exosomes from bovine serum at 0, 14, and 21 days of early pregnancy and analyzed the expression profile of exosomal miRNAs through RNA-seq technology. We identified 472 miRNA precursor sequences and 367 mature miRNA sequences in the three sample groups, with the majority of the miRNAs having high abundance. Differentially expressed miRNAs (DEmiRNAs) were screened, and 20 DEmiRNAs were obtained. The differential expression analysis results show that compared to day 0, there were 15 DEmiRNAs in the serum on day 14 and 5 on day 21 of pregnancy. Compared to the 14th day of pregnancy, there were eight DEmiRNAs in the serum on the 21st day of pregnancy. Bioinformatics analysis shows that the target genes of DEmiRNAs regulated the signaling pathways closely related to early pregnancy, including the VEGF, NF-κB, and MAPK signaling pathways. In addition, the newly discovered miRNAs were bta-miR-3604, bta-miR-2889, bta-miR-3432a, and bta-miR-409b. These results provide a theoretical reference for screening the molecular markers for early pregnancy establishment and maternal recognition of pregnancy (MRP) in cattle and new ideas for shortening the calving interval in cows.

RNA-seq reveals the role of miR-223 in alleviating inflammation of bovine mammary epithelial cells.

Bovine mammary epithelial cells (bMECs) are involved in the early defense against the invasion of intramammary pathogens and are essential for the health of bovine mammary gland. MicroRNA (MiRNA) is a key factor that regulates cell state and physiological function. In the present study, the transcriptome profiles of miR-223 inhibitor transfection group (miR-223_Inhibitor) and negative control inhibitor transfection group (NC_Inhibitor) within bMECs were detected via the RNA sequencing (RNA-seq) platform. Based on these experiments, the differentially expressed mRNAs (DE-mRNAs) of the miR-223_Inhibitor transfection group were screened, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses of DE-mRNAs were performed. The results revealed that compared with the NC_Inhibitor, 224 differentially expressed genes (DEGs) were identified in the miR-223_Inhibitor, including 184 upregulated and 40 downregulated genes. The functional annotation of the above DEGs indicated that some of these genes are involved in the immune response generated by extracellular substance stimulation, regulation of the activity of cytokines and chemokines, and the immune signaling pathways of NF-κB and TNF. Meanwhile, miR-223_inhibitor upregulated the immune key genes IRF1 and NFκBIA, cytokines IL-6 and IL-24, as well as chemokines CXCL3, CXCL5, and CCR6, triggering a signaling cascade response that exacerbated inflammation in bMECs. These results suggested that miR-223 plays an important role in inhibiting the inflammatory response and maintaining the stability of bMECs, and is a potential target for treating mastitis in dairy cows.

Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells.

Bovine mammary epithelial cells (bMECs) are part of the first line of defense against pathogens. In recent studies, bta-miR-223 has been reported to activate congenital and innate immunity against inflammatory damage during the pathogenesis of mastitis in dairy cows. The purpose of this study was to identify the regulatory mechanism of bta-miR-223 and its downstream target genes in inflammatory bMECs. A double luciferase reporter gene assay demonstrated that ras homolog family member B (RHOB) was the target gene of bta-miR-223. To further elucidate the role of bta-miR-223 in congenital immune responses, bta-miR-223 mimics (mimic/inhibitor) were transfected into bMECs stimulated with lipopolysaccharide (LPS), which activates the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect secreted inflammatory factors. Results showed that bta-miR-223 expression during inflammation in bMECs reduced the secretion of inflammatory factors by targeting RHOB and deactivation of NF-κB gene activity. Silencing RHOB inhibited LPS-induced inflammatory response in bMECs. Overall, bta-miR-223 attenuated LPS-induced inflammatory response, and acted as a negative feedback regulator via targeting RHOB, providing a novel avenue for mastitis treatment.

Correction: Jiao et al. Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells. Cells 2022, 11, 3144.

In the original publication [...].

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway.

Mastitis is characterized by inflammatory damage to mammary gland tissue, which could decline milk production and quality and significantly affect the economic benefits of ranching. MicroRNAs (miRNAs), such as miR-199a-3p, are novel therapeutic targets in inflammation, and their regulation is an effective strategy for inflammation control. Despite its importance in humans and animals, the molecular mechanism of bovine miR-199a-3p (bta-miR-199a-3p) in dairy cow mastitis and bovine mammary epithelial cell (bMEC) inflammation is unclear. In our study, a bovine mammary epithelial cell line (MAC-T) induced by lipopolysaccharide (LPS) was used as an inflammatory cell model to investigate the molecular mechanism of bta-miR-199a-3p in the MAC-T inflammatory response. bta-miR-199a-3p was up-regulated in the LPS-induced MAC-T cells, while CD2-associated protein (CD2AP) was revealed as its target gene in a double luciferase reporter gene experiment. In addition, the overexpression of bta-miR-199a-3p negatively regulated the expression of CD2AP and the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa-B (NF-κB) signaling pathway. These subsequently inhibited the secretion of related inflammatory factors (TNF-α, IL-1ß, and IL-6) and the expression of apoptotic genes (CASP3 and CASP9), thereby alleviating the LPS-challenged inflammatory response in the MAC-T cells. Silencing of bta-miR-199a-3p, however, reversed the above effects. Thus, bta-miR-199a-3p inhibits LPS-induced inflammation in bMECs by directly targeting CD2AP and regulating the PI3K/AKT/NF-κB signaling pathway. This study reveals the potential regulatory mechanism of bta-miR-199a-3p in bMEC inflammatory immune response and may serve as a useful target for the treatment of mastitis.

Differential mRNA Expression Profiling Reveals the Role of MiR-375 in Inflammation of Bovine Mammary Epithelial Cells.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate post-transcriptional gene expression and several biological processes. Bovine mammary epithelial cells (bMECs) mediate critical immune responses in the mammary gland and the occurrence of mastitis. Current research focuses on miRNA regulation of bMECs, but the miR-375 regulatory mechanism in bMECs is unclear. This study explored the role of miR-375 by profiling the transcriptome of miR-375-silenced bMECs using RNA-seq and identifying differentially expressed mRNAs (DIE-mRNAs). There were 63 DIE-mRNAs, including 48 down-regulated and 15 up-regulated mRNAs between miR-375-silenced bMECs and the controls. The Kyoto encyclopedia of genes and genomes (KEGG) and Gene Ontology (GO) functional analysis showed that the DIE-mRNAs enriched nuclear receptor subfamily 4 group A member 1 (NR4A1) and protein tyrosine phosphatase non-receptor type 5 (PTPN5) anti-inflammatory genes of the mitogen-activated protein kinase (MAPK) signaling pathway. However, they showed an opposite trend to the expression of miR-375 silencing, suggesting that miR-375 promotes bMEC inflammation through the MAPK signaling pathway. The findings of this study provide a new reference for understanding the regulation of bMEC inflammation and cow mastitis.

Role of Long Noncoding RNAs in the Regulation of Cellular Immune Response and Inflammatory Diseases.

Long noncoding RNAs (lncRNAs) are recently discovered genetic regulatory molecules that regulate immune responses and are closely associated with the occurrence and development of various diseases, including inflammation, in humans and animals. Under specific physiological conditions, lncRNA expression varies at the cell or tissue level, and lncRNAs can bind to specific miRNAs, target mRNAs, and target proteins to participate in certain processes, such as cell differentiation and inflammatory responses, via the corresponding signaling pathways. This review article summarizes the regulatory role of lncRNAs in macrophage polarization, dendritic cell differentiation, T cell differentiation, and endothelial and epithelial inflammation. In addition, it describes the molecular mechanism of lncRNAs in acute kidney injury, hepatitis, inflammatory injury of the lung, osteoarthritis, mastitis, and neuroinflammation to provide a reference for the molecular regulatory network as well as the genetic diagnosis and treatment of inflammatory diseases in humans and animals.

Progress in Expression Pattern and Molecular Regulation Mechanism of LncRNA in Bovine Mastitis.

Bovine mastitis is an inflammatory disease caused by pathogenic microbial infection, trauma, or other factors. Its morbidity is high, and it is difficult to cure, causing great harm to the health of cows and the safety of dairy products. Susceptibility or resistance to mastitis in individual cows is mainly determined by genetic factors, including coding genes and non-coding genes. Long non-coding RNAs (lncRNAs) are a class of endogenous non-coding RNA molecules with a length of more than 200 nucleotides (nt) that have recently been discovered. They can regulate the immune response of humans and animals on three levels (transcription, epigenetic modification, and post-transcription), and are widely involved in the pathological process of inflammatory diseases. Over the past few years, extensive findings revealed basic roles of lncRNAs in inflammation, especially bovine mastitis. This paper reviews the expression pattern and mechanism of long non-coding RNA (lncRNA) in inflammatory diseases, emphasizes on the latest research progress of the lncRNA expression pattern and molecular regulatory mechanism in bovine mastitis, analyzes the molecular regulatory network of differentially expressed lncRNAs, and looks forward to the research and application prospect of lncRNA in bovine mastitis, laying a foundation for molecular breeding and the biological therapy of bovine mastitis.